Particle-size reduction apparatus, and use thereof

ABSTRACT

A sterilisable particle-size reduction apparatus, component parts thereof and a method of sterilising and validating sterility thereof are provided. Also provided is use thereof to prepare sterile suspensions of drugs.

This invention relates to a particle-size reduction apparatus,sterilisation thereof and use thereof to prepare suspensions of drugs,in particular for administration via nebulizers.

Previously it was acceptable for drugs intended for use in nebulizers tobe prepared under “clean” conditions. Recently, however, suchformulations have caused problems in the US due to contamination, andthe US FDA has implemented a requirement for all nebulizer solutions tobe sterile. In the light of the US FDA decision it is necessary toproduce sterile suspension drugs in the US.

The sterilisation of suspensions raises particular problems. Thestandard means of sterilisation—that is, the raising of the temperatureof the formulation to 121° C. for 15 minutes—frequently destroys one ormore of the components of the formulation, so only chemicallythermostable products can be sterilised by this method. The desiredbiological activity of the formulation commonly requires that the massmedian diameter of particles of the drug lie within a narrow range(average diameter typically less than 5 μm). End sterilisation may alterparticle size. In addition this treatment results in the clumping oragglomeration of the drug particles in the suspension such that theefficacy of the resulting product is impaired or abolished.

Known alternative methods for the sterilisation of pharmaceuticals areinappropriate for sterilising suspension formulations of drugs.Solutions of pharmaceuticals may be sterilised by passage though afilter having a pore size of not more than 0.2 μm. However this cannotbe used in the case of suspensions as the required particle size inthese formulations (typically 2-5 μm) is significantly greater than thisfilter pore size. Similarly, pharmaceuticals may generally be sterilisedby gamma-irradiation, but Budesonide, for example, is destroyed by suchtreatment (see for example, WO 00/25745). Cold sterilisation usingethylene oxide and carbon dioxide is also known, but stability ofBudesonide under these sterilisation conditions has yet to bedemonstrated. No further methods for the sterilisation ofpharmaceuticals are currently acceptable to regulatory agencies.

Drugs typically provided as nebule suspensions are the steroidsFluticasone and Budesonide, which are used to treat asthma and chronicobstructive pulmonary disorder. These drugs are very insoluble in waterand are sold as non-sterile powders.

A method of sterilising dry, powdered Budesonide is known from WO99/25359. This method of sterilisation is, however, problematic as itrequires Budesonide powder to be sterilised and then mixed with theother components of the formulation under sterile conditions. The drugformulation is subsequently prepared under sterile conditions.

International Application No. PCT/GB03/00702 (incorporated herein byreference) describes a solvent based sterilisation method forsterilising pharmaceuticals, in particular suspensions of drugs for usein nebulizers. A sterile composition of a pharmaceutical compound isprepared by combining solvent with a non-sterile pharmaceutical compoundto form a solution, and then filtering the solution to yield a sterilepharmaceutical compound. All or part of the solvent is optionallyremoved to form a suspension, and under sterile conditions the compoundis combined with a pharmaceutically acceptable carrier.

In order to be effective in the lungs, the particle size of an activeingredient in a suspension must be within a certain size range—typicallythe mass median diameter of the particles in the suspension is less than10 μm. The sterile suspension may, therefore, be passed through aparticle-size reduction apparatus, such as a homogenizer,Microfluidizer® or similar device to reduce the average mass mediandiameter of the particles.

A suitable device, referred to as a Microfluidizer®, is available fromMicrofluidics, Inc. (MFIC), described in WO 99/07466 (incorporatedherein by reference). Examples of Microfluidizer® apparatus suitable forproduction scale particle-size reduction of a pharmaceutical suspensioninclude the M-610 and M-210EH series machines. However, these devicescannot be sterilised.

Particle-size reduction apparatus such as the Microfluidizer® apparatustypically operate under high pressures and comprise a plunger and a sealto separate the high pressure end of the apparatus from the low pressureend.

It is extremely important that the plunger seal maintains its integritythroughout the particle-size reduction process because if the seal wereto fail, the sterility of the process could be compromised. The seal istherefore a high maintenance component that needs to be regularlyremoved for inspection and/or replaced.

Prior art apparatus is routinely supplied with more than one (most oftentwo) interaction chambers arranged in series; with the first interactionchamber having internal conduits of the smallest size, having a circularcross-section with a diameter in the range from 10 μm, preferably 30 μmto 150 μm, more preferably to 100 μm; and the second interaction chamberhaving internal conduits of larger size, having a circular cross-sectionwith a diameter in the range from 200 μm, preferably 300 μm to 600 μm,more preferably to 500 μm. For example, the M-120EH machine is suppliedwith interaction chambers in which the first chamber has conduits withdimensions down to approximately 78 μm and the second chamber withdimensions down to approximately 400 μm.

Our co-pending application (International Application No.PCT/GB04/03574), addresses some of the disadvantages discussed above. Inparticular, a sterilisable particle-size reduction apparatus isdescribed, along with component parts that help sterilisation to beachieved. This apparatus can, therefore, be used for the production of asterile suspension of a drug, such as Budesonide, for use in anebulizer.

However, the apparatus described in PCT/GB04/03574 and other prior artapparatus have the disadvantage that they can become blocked, forexample, by particles of the suspended material. Particularlysusceptible to becoming blocked are component parts that contain flowpassages/conduits having relatively small transverse cross-sectionalarea, such as an interaction chamber, which is used to reduce the sizeof particles in a suspension (described in more detail later).

For example, in order to achieve a sufficient degree of comminution andan appropriate size distribution and morphology, the recommendedparticle-size reduction method using the prior art Microfluidizer®apparatus employs an interaction chamber having three 87 μm diameter,circular flow passages (i.e. having a transverse cross-sectional area ofapproximately 5.9×10³ μm²).

It is important to minimise the possibility of a particle-size reductionapparatus becoming blocked because blockages can be difficult to detect,especially when using interaction chambers containing more than one flowpassage, and blockages can prevent the complete sterilisation of theapparatus.

A further disadvantage of prior art methods that employ relatively smalldiameter flow passages, e.g. 87 μm diameter, circular flow passages asdiscussed above; is that such methods optimally require the use of highpressure pumps (generating up to 210 MPa [30,000 psi]), to forcesuspension around the apparatus. Such high-pressure pumps can bedifficult to sterilise.

It is an object of the invention to overcome or at least ameliorateproblems associated with the prior art apparatus.

It has now surprisingly been found that an acceptable reduction inparticle size, as well as a suitable particle size distribution may beachieved using one or more interaction chambers having flow passagesthat are substantially larger than was hitherto considered to benecessary.

The present invention thus provides a method of producing a comminutedsuspension of particles, which comprises:

-   -   subjecting a suspension of particles to a comminution procedure        carried out in a sterilised particle-size reduction apparatus;        -   said particle size reduction apparatus comprising at least            one interaction chamber for reducing the particle size of            the suspension, the or each interaction chamber being            provided with a flow passage through which the suspension is            forced, and an intensifier for forcing the suspension            through the flow passage of the interaction chamber or            interaction chambers, and    -   recovering a suspension of particles of reduced size;

characterised in that the transverse cross-sectional area of said flowpassage is not less than 3.1×10⁴ μm².

In a preferred embodiment of the invention the transversecross-sectional area of the flow passage is in the range of 3.1×10⁴ to2.8×10⁵ μm². More preferably, the transverse cross-sectional area of theflow passage is in the range of 4.9×10⁴ to 2.0×10⁵ μm², and mostpreferably in the range of 7.1×10⁴ to 1.3×10⁵ μm².

A further advantage of the method of the invention is that it can becarried out at lower pressure than typical particle-size reductionmethods using prior art apparatus, which operate at “high pressure” (upto 210 MPa [30,000 psi]). Accordingly, there is provided a method ofproducing a comminuted suspension of particles, wherein a suspension ofparticles is forced from the intensifier at a pressure not exceeding 69MPa (10,000 psi). More preferably, the pressure at which suspension isforced from the intensifier is in the range of 21-48 MPa (3,000-7,000psi), still more preferably in the range of 28-41 MPa (4,000-6,000 psi),and most preferably at a pressure of approximately 34 MPa (5,000 psi).

The method of the invention can comprise multiple rounds of comminutionin order to produce a comminuted suspension of particles of the desiredsize. Accordingly, there is provided a method of producing a comminutedsuspension of particles, which comprises:

-   -   (a) subjecting a suspension of particles to a comminution        procedure carried out in a sterilised particle-size reduction        apparatus; said particle-size reduction apparatus comprising at        least one interaction chamber for reducing the particle size of        the suspension, the or each interaction chamber being provided        with a flow passage through which the suspension is forced, and        an intensifier for forcing the suspension through the flow        passage of the interaction chamber or interaction chambers; to        obtain a comminuted suspension of particles; characterised in        that the transverse cross-sectional area of said flow passage is        not less than 3.1×10⁴ μm²;    -   (b) optionally recovering a comminuted suspension of particles        from step (a);    -   (c) subjecting a comminuted suspension of particles from        step (a) to at least one further comminution procedure carried        out in a sterilised particle-size reduction apparatus as defined        in part (a); and    -   (d) recovering a comminuted suspension of particles of reduced        size.

In preferred embodiments of the above method up to 50 comminutionprocedures are carried out. More preferably, the number of comminutionprocedures carried out is in the range of 10 to 50, 14 to 40 and 20 to30.

In order to determine when particles of the desired size have beenproduced, there is provided a further embodiment of the above method,which comprises: recovering a comminuted suspension of particles afterone or more further comminution procedures, measuring the size ofrecovered particles, and on the basis of the measured sizes, subjectingthe suspension to one or more further comminution procedures, ifnecessary.

The above methods are suitable for producing a comminuted suspension ofparticles. Typically, the mass median diameter of particles in therecovered suspension of particles is in the range of 1-10 μm, preferablyin the range of 1-5 μm and more preferably, the mass median diameter ofparticles in the recovered suspension of particles is in the range of2-3 μm.

The invention further provides a modification of the above methodswherein the interaction chamber and the intensifier are integrallycombined into a pump.

In accordance with a further object of the invention, there is alsoprovided a sterilisable particle-size reduction apparatus. Saidsterilisable particle-size reduction apparatus, comprising:

-   -   at least one interaction chamber for reducing the particle size        of the suspension, the or each interaction chamber being        provided with a flow passage through which the suspension is        forced; and    -   an intensifier for forcing the suspension through the flow        passage of the interaction chamber or interaction chambers;

characterised in that the transverse cross-sectional area of said flowpassage is not less than 3.1×10⁴ μm².

Although interaction chambers of the sterilisable particle-sizereduction apparatus may be arranged in any suitable combination, e.g. inparallel or in series; a preferred sterilisable particle-size reductionapparatus of the invention comprises from 1 to 4 interaction chambersarranged in series. More preferably, the apparatus comprises a first anda second interaction chamber arranged in series.

Furthermore, while each interaction chamber may be provided with one ormore flow passages/conduits e.g. 1, 2, 3, 4 or 5; in accordance with apreferred embodiment, the interaction chambers are provided with asingle flow passage. Such an arrangement has the advantage that anyblockages that may occur can be more easily detected.

Thus, a preferred sterilisable particle-size reduction apparatuscomprises a first and a second interaction chamber arranged in series,wherein each interaction chamber is provided with a single flow passage.

In certain embodiments of the invention, the transverse cross-sectionalarea of the flow passage(s) of said first interaction chamber isapproximately the same as the transverse cross-sectional area of theflow passage(s) of said second interaction chamber. Preferably, however,the transverse cross-sectional area of the flow passage(s) of said firstinteraction chamber is greater than the transverse cross-sectional areaof the flow passage(s) of said second interaction chamber. In a morepreferred embodiment, the transverse cross-sectional area of the flowpassage of said first interaction chamber is approximately 1.3×10⁵ μm²,and the transverse cross-sectional area of the flow passage of saidsecond interaction chamber is approximately 7.1×10⁴ μm².

Typically, the flow passage/conduit is circular in cross-section.Accordingly, said flow passage/passages preferably have a maximumtransverse diameter which is not less than 200 μm; more preferably themaximum transverse diameter is in the range of 200-600 μm; still morepreferably in the range of 250-500 μm; and most preferably in the rangeof 300-400 μm.

The particle-size reduction apparatus may be any device that achievesreduction of the mass median diameter of particles in a suspension. In aparticular embodiment, the apparatus is a Microfluidizer® —suitablymodel M-110, M-610, or M-210EH, adapted according to the invention to besterilisable.

Particular adaptations are set out below, and described in more detailin a specific embodiment of the invention. In general, to besterilisable, apparatus of the invention comprise at least one,preferably two or more of the following features:

-   -   (1) there is no conduit between the output and input of the        intensifier other than via the interaction chamber;    -   (2) valves in conduits between the intensifier and the        interaction chamber are diaphragm needle valves;    -   (3) non-return valves in the apparatus have metal-to-metal        seats;    -   (4) the plunger seal in the intensifier is adapted to be        sterilised;    -   (5) the bushing assembly in the intensifier allows access of        sterilising steam or water to the plunger seal;    -   (6) the cam nut in the intensifier is adapted to be sterilised;    -   (7) a rupture disc is used as a pressure relief valve; and    -   (8) a seal is provided to prevent suspension from reaching the        driving fluid that drives the intensifier in the event of        failure of the plunger seal.

By “sterilisable” it is meant that sterility sufficient to satisfy MCAand FDA regulations for pharmaceutical use is achieved. By way ofexample, at the present time, the MCA requires a 6-log reduction insuitably heat-resistant bacterial spores (e.g. Geobacillusstearothermophilus, ATCC No. 7953) to be demonstrated—that is, thenumber of spores present after sterilisation is reduced by 6 log incomparison to the number of spores present before sterilisation. In oneembodiment, to demonstrate sterilisation, a challenge of heat-resistantbacterial spores in excess of 1 million is administered and thensterilisation carried out. If total kill of spores is demonstrated thensterilisation has been achieved. The FDA may allow an extrapolation ofsterility from a short time period. Hence, if a 3-log reduction isdemonstrated in x minutes then the FDA may allow an extrapolation to a6-log reduction in 2x minutes.

By “high pressure” it is meant pressures in excess of 69 MPa (10,000psi), preferably in excess of 138 MPa (20,000 psi) and more preferablyup to around 217 MPa (30,000 psi). Prior art apparatus typically operateusing oil at a pressure of up to 34 MPa (5,000 psi) to drive a piston inthe intensifier, resulting in a pressure in the plunger barrel of theintensifier of up to 217 MPa (30,000 psi). Hence, suspension exits theplunger barrel of the intensifier at this pressure and is directed tothe interaction chamber or chambers. On exiting the last interactionchamber the pressure of the suspension has typically reduced to belowabout 0.69 MPa (100 psi).

The apparatus of the present invention can operate at far lowerpressures than the apparatus of the prior art. For example, in apreferred embodiment, suspension exits the plunger barrel of theintensifier at a pressure of 34 MPa (5,000 psi). However, even at suchlow pressures, due to the relatively large flow passage(s) of theapparatus of the invention, a greater volume of suspension can beprocessed in a defined period of time, than can be processed byapparatus and methods of the prior art, that require far higherpressure, e.g. up to 217 MPa (30,000 psi).

For example, a sterilisable particle-size reduction apparatus of theinvention, which comprises a single interaction chamber having a singleflow passage with a circular cross-section of diameter 400 μm, canprocess approximately 1600 ml/min of suspension at 34 MPa (5,000 psi).Therefore, a typical batch of 12 litres of suspension can be subjectedto 20 rounds of comminution in 150 minutes.

A suitable pump for use in the apparatus and methods of the presentinvention is a diaphragm pump. An advantage of using a diaphragm pump isthat it can be more easily sterilised than a high-pressure pump.

The intensifier suitably comprises an output and an input, and theinteraction chamber comprises an input and an output, the output of theintensifier being connected to the input of the interaction chamber andthe output of the interaction chamber being connected to the input ofthe intensifier, and there is no conduit between the output of theintensifier and the input of the intensifier other than via theinteraction chamber. This means that all the suspension leaving theintensifier at must travel through the interaction chamber, in whichparticle-size reduction takes place, before exiting the apparatus. Inparticular this means that the sterilisable particle-size reductionapparatus of the present invention does not comprise a bypass line thatwould allow product (and sterilising steam or water) to bypass theinteraction chamber, as the presence of such a line means that thissection of the apparatus cannot be sterilised.

In the apparatus and methods of the present invention, it has been foundthat best results are achieved, in reducing the particle size of asuspension of Budesonide, when the suspension exiting the intensifierpasses first into a interaction chamber with larger flow passge/conduitsize and then into an interaction chamber with smaller flowpassage/conduit size.

The intensifier and interaction chamber(s) are linked by conduits, andthe conduits are generally provided with a number of valves to controlor direct flow of material. In one embodiment, the valves in theconduits between the intensifier and the interaction chamber aresterilisable diaphragm needle valves. Other valves in the apparatus arenon-return valves, which prevent flow of suspension in the wrongdirection—that is, the non-return valves ensure a flow of product in onedirection from the intensifier to the interaction chamber. Preferably,the non-return valves in conduits between the intensifier and theinteraction chamber(s) have metal-to-metal seats. The provision ofmetal-to-metal seats enables effective sterilisation of the non-returnvalves in situ.

In particular apparatus, the intensifier comprises a bore and areciprocating plunger and a seal between the plunger and the bore. Thepurpose of the seal is to separate the higher pressure side of theintensifier from the lower pressure side. In prior art apparatus, theseal must be able to withstand high pressures (up to 210 MPa [30,000psi]), without extruding or otherwise failing. This is not such animportant factor in the apparatus and methods of the present invention.Nevertheless, a preferred seal, used in apparatus of the invention, isadapted to be sterilisable, preferably incorporating a brace to preventsides of the seal from collapsing, which brace is made of or comprises aresilient plastics material. The seal is described in more detail below.

In other particular apparatus, the intensifier comprises a reciprocatingplunger and a bushing assembly to guide the plunger as it reciprocateswithin the plunger chamber or barrel. The bushing assembly preferablycomprises a bushing holder and a bushing supported within the bushingholder. This bushing assembly preferably comprises a channel in or onthe surface of the bushing assembly, to allow sterilising steam or waterto pass through the bushing assembly whilst the plunger is in place. Thechannel in or on the surface of the bushing assembly may typically be agroove or a conduit, and may be located on the outer or inner surface ofthe bushing and/or on the bushing holder. The groove or conduit may beof any reasonable dimensions and there may be any number of grooves orconduits, enabling steam or water to pass through the bushing assemblywhilst the plunger is in place. This bushing assembly means thatsterilising steam or sterilising water has access through the bushing tocomponents of the apparatus that would otherwise be difficult orimpossible to sterilise, and this arrangement especially allows accessof sterilising water or steam to the back of the plunger seal.

Referring to the apparatus in the figures, one end of the intensifierplunger is connected via a threaded cam nut to a connecting rod having ascrew thread to receive the cam nut. The dimensions of the screw threadand the thread of the cam nut are such that as the nut is screwed ontothe connecting rod (con rod), respective mating surfaces on the cam nutand the con rod mate simultaneously, which avoids nooks and cranniesthat may harbour microorganisms and thus renders this portion of theapparatus sterilisable. The plunger in use bears on the front end of thecon rod and is held loosely in place by the cam nut. As the plunger isdriven in one direction, the cam nut approaches and then hits andtriggers an air switch, changing the direction of flow of oil from oillines to the piston around the con rod and sending the plunger back inthe reverse direction.

Optionally, a heat exchanger is provided to control the temperature ofthe suspension and preferably to maintain it at from 7° C. to 40° C. inuse. If the suspension is a drug suspension, it is important to maintainthe temperature within a certain range because some drugs aresusceptible to heat degradation. By way of example, Budesonide may bedegraded by long exposure to temperatures above 40° C., so duringBudesonide processing the temperature is preferably maintained below 50°C., more preferably below 40° C. The apparatus and methods of thepresent invention have a further advantage that heating of the apparatusduring the comminution procedure is greatly reduced from that of priorart methods and therefore, use of a heat exchanger during comminutionmay not be necessary.

A further use of the heat exchanger is during sterilisation of theapparatus. Time is spent heating various components of the apparatus upto the sterilising temperature. Therefore, in a preferred method ofsterilisation, the heat exchanger is used to heat the interactionchamber or chambers, and preferably also the piping immediatelysurrounding the chambers, to reduce the time required for theinteraction chambers to reach the required temperature. In a furtherpreferred embodiment, the apparatus comprises a first heat exchanger tomaintain the temperature of the suspension in the interaction chamberand a second heat exchanger to maintain the temperature of thesuspension in the intensifier, wherein the first and second heatexchangers are independently controlled.

The apparatus optionally comprises at least one pressure relief valve,so that if excessive pressure builds up on the low pressure side of theapparatus, that is to say downstream of the interaction chamber, thispressure can be relieved instead of leading to damage of the lowpressure side. The valve is preferably a rupture disc. By rupture discit is meant a valve that bursts if the pressure at the valve exceeds acertain value. Hence, the rupture disc acts as a safety mechanism, toalert an operator to the fact that a pressure exceeding the specifiedvalue has been reached at that point in the apparatus. This couldtypically occur if one of the non-return valves of the apparatus hasfailed or if there is a blockage in the return line. In one embodiment,the rupture disc will burst if the pressure at the disc exceeds 150 psi.In another embodiment, the rupture disc is positioned so as to preventdamage to the interaction chamber and associated pipework and valvesshould the plunger seal fail.

During operation of the apparatus, once the apparatus has beensterilised it is used to reduce the particle size, of a sterilesuspension. If there were to be a failure, possibly a transient failure,leading to excess pressure on the low-pressure side of the apparatusthen rupture of the disc alerts the operator to the failure. In thisevent, the suspension in the apparatus is then discarded, as the failurecould lead to contamination, and the risk of producing a non-sterilesuspension. Hence, an advantage of using this rupture disc is that atransient failure, which in the art would be accommodated by transientopening and closing of a standard relief valve, does not mask a failureof sterility in the apparatus and hence in the suspension beingprocessed.

Particular apparatus further comprise a seal that prevents suspensionfrom reaching the driving fluid that drives the intensifier in the eventof failure of the plunger seal. It is advantageous to prevent suspensionfrom interfering with the hydraulic pump section of the apparatus if theplunger seal fails. This seal is typically capable of withstandingpressures of 1 MPa (150 psi) at 200° C. while the plunger is moving.Preferably, this seal is a lip-type seal and is manufactured from PTFE.The seal may further comprise a coiled metal support inner spring tohelp avoid collapse, extrusion or distortion at high temperature.

In an example of using the apparatus, product is processed in severalcycles. In each cycle, product is passed from a feed tank into theparticle-size reduction apparatus. As the cycle progresses, productaccumulates in a recycle tank. Once the feed tank is empty or nearlyempty, a cycle is deemed to be finished, and the feed tank is thenre-filled from the recycle tank, indicating that a further cycle isbeginning. We have circulated a suspension of Budesonide in water andTween up to 50 times at 34 MPa [5,000 psi] (depending on the selectionand arrangement of interaction chambers used), in order to achieve adesired particle size distribution of 2-3 μm. It is possible tocirculate the suspension with the apparatus operating at lower or higherpressure (e.g. 7-69 MPa [1,000-10,000 psi]), in which case a larger orsmaller number of cycles, respectively, would be required to achieve thesame particle size distribution for a given combination of interactionchambers.

The apparatus of the present invention may comprise modified components,as described in our co-pending patent application (InternationalApplication No. PCT/GB04/03574), which is incorporated herein byreference. For example, one such useful component is a modified bushingassembly for use with a cylindrical plunger, comprising a bushing holderand a bushing, held in place by the bushing holder, wherein the bushingassembly comprises one or more conduits to allow passage of sterilisingsteam or water therethrough.

International Application No. PCT/GB04/03574 also provides a bushingassembly for a plunger that reciprocates in a plunger barrel, comprisinga bushing holder which attaches to a neck of the barrel and a bushingheld in situ by the bushing holder and which guides the plunger into andout of the barrel, wherein the bushing and/or the bushing holdercomprises one or more conduits to allow passage of sterilising steam orwater through the bushing assembly.

During sterilisation of the apparatus, the conduits allow access ofsterilising water or steam to parts of the apparatus that mightotherwise be difficult or impossible to sterilise. In particular,sterilising water or steam can now have access to the plunger seal.During sterilisation, sterilising water or steam passes through thebushing assembly and sterilises the back of the plunger seal. Usually,whilst sterilisation is taking place, the apparatus is run at a reducedrate, enabling sterilisation of all parts of the intensifier, both thehigh-pressure side and the low-pressure side, the high-pressure sidebeing sterilised by steam introduced directly into the plunger barrel.The plunger barrel may, for instance, be the plunger barrel of aparticle-size reduction apparatus, such as a Microfluidizer®.

By conduits with respect to the bushing holder described above, it ismeant grooves, channels or the like through which the steam or water maypass. The grooves or channels may be of any reasonable dimensions, solong as passage of the steam or water therethrough is permitted.

Said grooves/channels may be located anywhere on the outer or innersurface of the bushing and may be aligned in any direction, so long asthey permit passage of steam or water through the bushing assembly. Forexample, the bushing may comprise one or more grooves located on itsouter surface. Alternatively, or in addition, said bushing may compriseone or more grooves located on its inner surface. The grooves may beparallel to the longitudinal axis of the bushing or said grooves may beformed in a spiral around the longitudinal axis of the bushing.

It is an option for the bushing assembly to comprise a bushing whichcomprises one or more grooves and a bushing holder which comprises oneor more grooves or one or more conduits to allow passage of steam orwater therethrough.

Where both the bushing and the bushing holder comprise one or moregrooves, it is preferred that said one or more grooves of said bushingand bushing holder are in alignment as this enables unhindered passageof steam through the bushing apparatus. Alignment of said one or moregrooves of the bushing and the bushing holder can be achieved using abushing assembly wherein said bushing further comprises one or moreprojections that cooperate with one or more recesses in said bushingholder in order to align said one or more grooves of said bushing withthose of the bushing holder. Alternatively said bushing holder has oneor more projections that cooperate with one or more recesses in thebushing.

Co-pending patent application (International Application No.PCT/GB04/03574) also provides an annular high-pressure seal that may beused in the apparatus of the present invention. This high-pressure sealfor a plunger reciprocating within a barrel, comprises lower and upperbody portions, said upper portion being in the form of a cup and havingsides surrounding a recess, the sides being outwardly deformable so thatrespective outer and inner edges of the sides of the cup make, in use,sealing contact with respectively the barrel and the plunger. The sealfurther comprising a brace to prevent the sides from collapsing into therecess under low pressure and wherein the brace comprises a resilientplastics material. This “high-pressure seal” is capable of withstandingpressures typically encountered in a particle-size reduction apparatus.Typically, a high-pressure seal can withstand pressures of up to 34 MPa(5,000 psi), preferably up to 69 MPa (10,000 psi), more preferably up to138 MPa (20,000 psi), and still more preferably, up to 210 MPa (30,000psi). Such a high-pressure seal is also sterilisable.

By “sterilisable” it is meant that sterility sufficient to satisfy MCAand FDA regulations for pharmaceutical use (as outlined above withrelation to sterility of the particle-size reduction apparatus) isachieved.

The seal employed in the apparatus and methods of the present invention(described in International Application No. PCT/GB04/03574), confers theadvantage that it can be sterilised, an especially important feature asthe seal comes into contact during operation of the apparatus withsuspension on the high-pressure side of the apparatus. In comparison,some prior art seals contain structural and surface features thatharbour microorganisms, rendering such seals incapable of sterilisation,and these features are avoided in the seal used in the invention.

The brace of the plunger seal presents a smooth surface free fromcavities. By free from cavities it is meant free from holes, cracks,gaps or other spaces in the otherwise solid mass of the brace.Minimising (and preferably eliminating) cavities in which microorganismsmay collect, ensures that complete sterilisation of the seal can takeplace.

The resilient plastics material of the brace is disposed in the recessbetween the cup sides of the plunger seal. The plastics material canfill the recess of the plunger seal so that the upper surface of saidplastics material is level with or nearly level with the height of thecup sides, i.e. the upper surface of said plastics material reaches atleast two thirds the height of the cup sides.

The plunger seal may further comprise a metal spring; if so this ispreferably enclosed within the resilient plastics material of the brace.Using a metal spring adds further strength or resilience to the brace ofthe seal, and enables choice of alternative plastic materials for thebrace.

Usually, the plunger seal is operable at temperatures up to 75° C.,preferably at temperatures up to 90° C., most preferably at temperaturesrequired for sterilisation of the apparatus, generally up to about 122°C. The plunger seal material may be virgin PTFE or glass-strengthenedPTFE. These materials are known to be capable of withstanding highpressures and temperatures without extruding. An example ofglass-strengthened PTFE from which seals of the invention can be made isRulon®.

It is preferred that the plunger seal brace is manufactured from adifferent material to that of the other seal components, so that the cupsides of the seal will deform outwardly under the pressures experiencedduring operation of the apparatus and form sealing contact with theplunger and the bore, but under low pressure, e.g. whilst the machine isat rest, the cup sides do not collapse inwardly leading to subsequentseal failure. The resilient plastics material of the brace is preferablymore flexible than the material of the upper and lower body portions ofthe seal. It is, however, an option for the brace to be manufacturedfrom the same material to that of the other seal components, so long asthe seal remains outwardly deformable in use. Preferred apparatus foruse in the methods of the invention, and component parts therefor, aresubstantially free of niches which can harbour microorganisms and/ortheir spores or which can shield them from the effects of thesterilising steam and/or water during sterilisation of the apparatus andits parts. For example, the apparatus preferably avoids unnecessarypipework or pipework containing dead-ends or inaccessible spaces thatwould represent such niches and compromise sterility or validationthereof.

The present invention further provides methods of sterilising aparticle-size reduction apparatus. A first method comprises the step ofcharging the particle-size reduction apparatus of the invention withsteam, to achieve sterilisation.

A sterilisation protocol may optionally be followed by a method ofvalidating sterilisation—in order to ensure that the sterilisation iseffective and/or complete.

In a particular embodiment of the present invention, sterilisation isdeemed to have occurred when a protocol, previously demonstrated toachieve a 6-log reduction in heat resistant bacterial spores isfollowed.

Generally, validation of sterility is carried out in order to establisha protocol that is demonstrated to result in a sterile apparatus, whichapparatus is then used to reduce the particle size of a sterilesuspension. Validation of sterility is not then routinely carried outwith every batch, but may be used as part of regular maintenance of theapparatus or to carry out spot checks on individual batches ofsuspension.

When sterilising the particle-size apparatus using steam, it has beenfound advantageous to insulate the valves and conduits downstream of theinteraction chamber, so as to maintain steam temperature duringsterilisation. Loss of heat from the steam can cause undesirablecondensation and loss of effective sterilisation.

Referring to a specific embodiment of the invention, described in moredetail in the examples, steam traps are used around the apparatus,located in places where condensate would develop and risk accumulating.The steam traps are open when the temperature is below 121° C. butduring sterilisation the traps are open until they have reached thesterilising temperature, generally 121° C., at which point they close.If the temperature in a trap drops, for example due to accumulation ofcondensate, the trap opens, releasing the condensate from the apparatus,and then will close again when the temperature has reached 121° C. Thusduring sterilisation, traps are continually opening and closing.

Temperature probes are used all around the apparatus to provide atemperature map of the apparatus and to confirm that the temperature inall relevant places is at least 121° C. The probes are connected to acentral monitoring unit, so that the duration of the sterilisationprocedure is timed from the point at which all relevant parts of themachine have reached the sterilising temperature.

During sterilisation the following steps are typically carried out:

-   -   steam traps are connected;    -   temperature monitors are connected;    -   steam is introduced into the apparatus, optionally with the        apparatus running;    -   temperature is monitored at each monitor until all have reached        the sterilising temperature, generally 121° C.;    -   during this period, the steam traps start in the open position        but close as they reach 121° C., opening and closing as        described above;    -   the time at which temperature recorded by each of the        temperature monitors has reached the sterilising temperature is        noted;    -   once all monitors have reached 121° C. then the sterilisation is        continued by continuing to introduce steam into the apparatus        for a predetermined period of time, this time being determined        empirically.

The number of steam traps connected to the apparatus varies with thetype of apparatus and depends on the particular sterilisation protocolbeing carried out. We have achieved good results using an M-210EHMicrofluidizer® with up to 20 steam traps, but it is an option to usefewer steam traps, for instance up to 10, but preferably at least 5steam traps are used.

The number of temperature monitors connected to the apparatus varieswith the type of apparatus used. We have achieved good results using anM-210EH Microfluidizer® with up to 10 temperature monitors, though it isan option to use fewer temperature monitors, for instance about 5temperature monitors, or more temperature monitors, for instance, up to20.

When the apparatus is allowed to run during introduction of steam, theapparatus is run at a slow speed. When an M-210EH Microfluidizer® isused, steam is introduced at a speed of typically up to half the runningspeed of the apparatus, and in some embodiments, up to a third of therunning speed of the apparatus.

In a particular embodiment, this period is determined by introducingheat resistant bacterial spores into the apparatus, introducing steaminto the apparatus and monitoring apparatus temperature until it hasreached the sterilising temperature; continuing to introduce steam for afirst known amount of time; determining whether after that first knownamount of time sterilisation has been achieved; and if sterilisation hasnot been achieved, repeating the method for a second, longer knownamount of time.

In practice, a protocol is determined that is accepted as ensuringsterilisation after a given period of time, and this time is noted and amargin of error, such as an additional at least 5, 10 or 20 per cent ofthe noted time, is added and this modified protocol is noted as thesterilising protocol. Also in practice, the intensifier tends to takelongest to reach an acceptable sterilising temperature. The intensifiercan be provided with a jacket or other insulation to help speed up thisprocess.

As the apparatus of the present invention tends to generate lessinternal heat than prior art apparatus a heat exchanger may additionallybe used to raise the temperature of the sterilising water or steam to anacceptable sterilising temperature.

During sterilisation, it is preferred that all steam exiting theintensifier passes through the interaction chambers—i.e. sterilisingsteam cannot bypass the interaction chambers, as this may risk creationof areas in the apparatus, around the chambers, which cannot besufficiently reached by the steam to achieve sterilisation. A jacket isalso optionally located around the interaction chambers. This jacket canbe used to increase the temperature of the interaction chambers usingsteam to assist sterilisation and it can be used to cool the interactionchambers when the machine is operated.

Whilst sterilising the apparatus described in the examples, as steam ispassed through the chambers it passes from a 3 mm diameter feed to a0.087 mm feed, potentially resulting in trapped condensation at theinteraction chamber exit. It is therefore preferred that steam isintroduced into the intensifier and, in addition, downstream of theinteraction chamber or chambers. This step assists in the rapidsterilisation of apparatus, conduits etc, located on the other side ofthe interaction chambers to the primary steam source. Pre-heating theinteraction chambers can also serve to reduce the potential problem oftrapped condensation at the interaction chamber exit.

A second method of sterilisation comprises charging the particle-sizereduction apparatus of the invention with pressurised, superheated waterso as to sterilise the apparatus.

When pressurised, superheated water is used for sterilisation, theintensifier can be operated so as to control the temperature of thewater during sterilisation. Operating the intensifier leads to anincrease in the pressure of the water within the apparatus, in turnleading to an increase in temperature which can be monitored. Hence, byadjusting pressure within the apparatus, temperature within theapparatus can also be adjusted and kept at or above a desiredsterilising temperature of 121° C. Following a preferred embodiment ofthe water-based sterilisation method, water is introduced into theapparatus at a temperature below 100° C., and this could suitably be atroom temperature, and the apparatus is then operated so as to increasethe water temperature up to the desired sterilising temperature.Temperature monitors located on the apparatus are used to confirm thatthe desired temperature has been reached, at which point sterilisationis continued at or above this temperature for a time period previouslydetermined to be accepted as resulting in sterilisation, this timeperiod being determined empirically.

When pressurised, superheated water is used for the sterilisationmethod, it is preferred that steam is nonetheless used for sterilisationof the isolation area of the intensifier, and the method comprisescharging the isolation area of the intensifier with steam, at atemperature the same as or higher than the temperature of the water,preferably at least 0.5° C. higher.

After sterilisation has been carried out, the water is cooled and, forexample, Budesonide suspension and optional extra ingredients such assurfactants are added. One option is to sterilise the apparatus usingsuper-heated water, then use sterile air to flush the system beforeintroducing a Budesonide suspension. Another option is to sterilise theapparatus using super-heated water containing surfactant, cool the waterand surfactant solution and then add the Budesonide suspension. In thisway, the end of the sterilising step becomes the beginning of thepriming step. Further, a filter can be used to collect microorganisms.

The present invention further provides a method of preparing a sterilesuspension, in particular a sterile suspension comprising Budesonide orFluticasone, comprising the steps of obtaining a sterile particle-sizereduction apparatus, passing a sterile suspension through the sterileapparatus, and monitoring particle size in the suspension. Preferably,the particle-size reduction apparatus is sterilised according to thesteam or water sterilisation methods of the present invention, asdescribed above. In one embodiment, particle size in the suspension ismonitored continuously as the suspension is passed through theapparatus. In another embodiment, particle size is monitored betweendiscrete passes. The suspension is passed through the apparatus untilthe desired final mass median diameter of the particles isobtained—typically 2-3 μm. Once the desired particle size has beenachieved, the sterile suspension may then be transferred from theapparatus to be packaged into sterile ampoules, preferably nebules.

In another aspect, the present invention also provides a sterile nebulecontaining a sterile suspension prepared according to the presentinvention. When the suspension in the nebule comprises Budesonide orFluticasone, the sterile nebule may be of use in the treatment of asthmaor chronic obstructive pulmonary disorder.

The sterility of components of the particle-size reduction apparatus ofthe invention can then be validated. For example, the sterility of abore may be validated by the following method, which is carried outunder sterile conditions. The method comprises the steps of removing aseal from the bore, under sterile conditions transferring the seal togrowth medium, observing whether there is growth of microorganisms inthe growth medium, calculating the number of microorganisms present, andthereby determining whether the bore is sterile. In a preferredembodiment, the method comprises the initial steps of inoculating theseal with a known quantity of heat-resistant bacterial spores, mostpreferably at least 1×10⁶ heat-resistant bacterial spores, inserting theseal into the bore, and carrying out a sterilisation protocol asdescribed above.

Sterility is judged according to the MCA and FDA guidelines. Thecomponent, such as a seal is typically incubated in the growth mediumunder conditions conducive to growth of microorganisms, and growth ofmicroorganisms indicates that the seal (and hence the bore) has not beensterilised effectively. In a preferred embodiment, the validation methodcomprises the steps of inserting a component of the apparatus, such as aseal, inoculated with a known number of heat resistant bacterial sporesinto the apparatus (e.g. into the bore), carrying out a procedureintended to sterilise the apparatus including the bore, and thenvalidating sterility of the component (e.g. the seal), and hence thebore or other component of the apparatus. The bore, for example, may bethe bore of a particle-size reducing apparatus and, in one embodiment;the sterility of the bore may be used as an indication of sterility ofthe entire apparatus.

The invention is now described in more detail with reference to theaccompanying drawings, in which:

FIG. 1 is a schematic diagram showing flow of suspension between thecomponent parts of a particle-size reduction apparatus;

FIGS. 2, 3 and 4, respectively are front, top and side views of aMicrofluidizer® M-210EH apparatus that can be modified in accordancewith the present invention;

FIG. 5 is a cross-sectional view of the intensifier of a Microfluidizer®M-210EH apparatus that can be modified in accordance with the presentinvention.

Referring to the drawings in more detail; FIG. 1 is a schematic diagramshowing flow of a sterile Budesonide suspension between the maincomponent parts of the particle-size reduction apparatus. The suspensionis generated in the reaction feed tank—by combining a sterile solutionof Budesonide in alcohol with an aqueous solution comprising Tween andwater. The sterile suspension is fed into the intensifier of theapparatus from the reaction feed tank via a conduit. The output from theintensifier leads, via a conduit, into the input of the interactionchamber. The interaction chamber has two outputs and hence, from theinteraction chamber, the suspension may follow either of two routes. Ifparticle size has been reduced to the desired final mass mediandiameter, the suspension leaves the apparatus for packaging in sterilecontainers, such as ampoules. If, however, particle size is still toolarge, the suspension leaves the interaction chamber and passes via aconduit into the recycling tank. The recycling tank then feeds thesuspension back into the reaction feed tank, from which the suspensionis fed back into the intensifier for another pass. Alternatively,product can be transferred from the recycling tank to be furtherprocessed and/or packaged.

As can be seen from FIG. 1, the suspension cannot pass from the outputof the intensifier to the input of the intensifier without passingthrough the interaction chamber, because there is no conduit between theoutput of the intensifier and the input of the intensifier other thanvia the interaction chamber.

In practice, the particle-size reduction apparatus is run in almostdiscrete passes. Suspension from the interaction chamber that must bepassed through the apparatus at least once more is fed into therecycling tank and accumulates there whilst the reaction feed tankempties. Only once the reaction feed tank is almost empty is suspensionfrom the recycling tank fed back into the reaction feed tank for anotherpass.

A Microfluidizer® M-210EH particle-size reduction apparatus (1) modifiedaccording to one embodiment of the present invention is now describedwith reference to FIGS. 2-5. FIG. 2 shows a front view of the modifiedapparatus, FIG. 3 shows a top view and FIG. 4 shows a left side view.

The Microfluidizer® comprises intensifier (13), interaction chambers (25and 26) and base unit (35) housing an oil tank, pump and motor (notshown).

Sterile suspension enters the Microfluidizer® from the reaction feedtank via input (3) and passes along conduit (5). At T-junction (7) theflow of suspension is split along two conduits (9 a and 9 b), which feedinto opposite ends of the symmetrical intensifier (13) via non-returnvalves (11 a and 11 b). The non-return valves prevent the suspensionfrom flowing back along conduits 9 a and 9 b, which might otherwiseresult due to the pressures created in the intensifier. Suspensionpasses into the plunger barrels (15 a and 15 b) at each end of theintensifier. Suspension is prevented from entering the isolationchambers (17 a and 17 b) by plunger seals (not shown on FIGS. 2-4).

Each M-210EH series machine contains an on-board 15 horsepowerelectric-hydraulic module within base unit (35) that provides power to adouble-acting intensifier plunger (not shown on FIGS. 2-4). Theintensifier plunger amplifies the hydraulic pressure and, in turn,imparts that pressure to the product stream. The intensifier typicallyhas a multiplier ratio of about 3:1 to 20:1. Process pressures rangingfrom 17 to 210 MPa (2,500 to 30,000 psi) may be selected. Preferably, aprocess pressure of approximately 5,000 psi is used. Therefore, thehydraulic module may be replaced by a diaphragm pump which can besterilised more easily.

The intensifier plunger supplies the desired pressure at a constant rateto the product stream. As the plunger travels in one direction, itdrives the suspension at constant pressure through the flow passage(s)in interaction chambers (25 and 26). As the intensifier plungercontinues its travel in one direction, a series of check valves allowsuspension to be drawn into the opposite end of the pump barrel. Oillines (31 and 33) provide a flow of oil within the plunger barrels thatregulates the direction of movement of the plunger within each plungerbarrel. Thus, as the intensifier plunger completes its stroke, itreverses direction and the new volume of suspension is pressurisedrepeating the process. This creates a constant flow of suspension atnear constant pressure through the interaction chamber.

Suspension at the desired pressure (e.g. 34 MPa [5,000 psi]) leaves eachplunger barrel 15 a and 15 b of the intensifier via non-return valves(19 a and 19 b) respectively, and passes along conduits (21 a and 21 brespectively). A pressure transducer (37) on conduit 21 b monitorspressure of the suspension as it passes along conduit 21 b. Once the twoflows of suspension along conduits 21 a and 21 b reach T-junction (22),the flows are combined in conduit (23).

From conduit (23), the pressurised suspension enters the interactionchambers (25 and 26) via diaphragm needle valve (24). It is within theinteraction chambers that particle-size reduction occurs, as thesuspension is forced through precisely defined fixed-geometrymicrochannels in the interaction chambers under the desired pressure(e.g. 34 MPa [5,000 psi]), creating shear and impact forces as theproduct stream impinges upon itself and on wear-resistant surfaces athigh velocities. The flow passage(s) in the first interaction chamber(25) have a transverse cross-sectional area of not less than 3.1×10⁴μm², and the flow passage(s) in the second interaction chamber (26) alsohave a transverse cross-sectional area of not less than 3.1×10⁴ μm², Thecombined forces of shear and impact within the microchannels act uponproducts to reduce mean particle size (mass mean diameter) and canreduce the mean particle size of a Budesonide suspension fromapproximately 50 μm to 2-3 μm in typically up to 50 passes through theMicrofluidizer® at 34 MPa (5,000 psi) However, more or less passesthrough the Microfluidizer® may be required, e.g. from 10 to 50 passes,or more than 50 passes, depending on the combination of interactionchambers selected for the particular apparatus.

Downstream of the interaction chambers there is a rupture disc (27),which bursts at 1 MPa (150 psi) in the event of a build up of pressurecaused by a blockage in the apparatus pipework.

Suspension leaves the interaction chamber via outlet (29). The outletmay be connected to a conduit for returning suspension that has not yetreached the desired particle size to the recycling tank (not shown)ready for another pass through the Microfluidizer®. The machine operatescomfortably at up to 1.6 litres per minute (depending on the particularcombination of interaction chamber(s) employed) at an operating pressureof typically 34 MPa (5,000 psi). A typical batch is 12 litres, and isgenerally passed up to 50 times through the apparatus.

When the mass median diameter of particles in the suspension has reachedthe desired particle size, the suspension may be fed from outlet (29)into the recycling tank before being diluted, mixed with otherexcipients and transferred to a means for sterile packaging (not shown),for example into sterile ampoules.

FIG. 5 shows a cross-section of the left hand side of the intensifierpart (100) of the modified apparatus. The following description of theleft-hand side of the intensifier applies also to the right-hand side,since the intensifier is symmetrical.

The intensifier comprises two main sections—plunger barrel (110) andisolation chamber (145). A plunger (115) is housed in the plunger barrel(110) and is connected via cam nut (135) to a connecting rod (140),which is located in the isolation chamber (145). The cam nut (135) isscrewed tightly onto the end of connecting rod (140) but plunger (115)is held loosely in position by cam nut (135).

Cam nut (135) interacts with an air switch [not shown but located in theposition surrounded by dotted lines (137)], which controls direction ofmovement of plunger (115) within plunger barrel (110). As plunger (115)is driven inwards within the plunger barrel, cam nut (135) approachesand then hits and triggers the air switch, changing the direction offlow of oil from the oil lines to the plunger around connecting rod(140) and forcing the plunger back in the reverse direction. The oilpressure used can be up to 34 MPa (5,000 psi), resulting in up to 210MPa (30,000 ps)i of pressure inside the plunger barrel. In the methodsof the present invention, the pressure inside the plunger barrel isgenerally selected to be approximately 34 MPa (5,000 psi), but may befrom 6.9-69 MPa (1,000-10,000 psi).

The plunger barrel is isolated from the isolation chamber via a plungerseal located in seal location (120). The plunger seal prevents flow ofsuspension from the plunger barrel to the isolation chamber in use andis designed to withstand high pressures (up to 210 MPa [30,000 psi]).

Between the plunger seal and the cam nut (135) is a bushing (130)supported within bushing housing (125). The bushing supports the plunger(115) as it reciprocates within the plunger barrel.

The back of the isolation chamber (145) is provided with two oppositelyfacing seals (150 and 155). Seal (155) retains oil used to drive theconnecting rod, whilst if there is any leakage of this oil the secondseal (150) ensures it passes into drain (160). The main purpose of seal(150), however, is to prevent suspension from interfering with thehydraulic pump section of the apparatus in the event of failure of theplunger seal. Seal (150) is a lip-type seal, made from PTFE, and iscapable of withstanding pressures of 1 MPa (150 psi) at 200° C. whilethe plunger is moving.

EXAMPLES Example 1 Sterilising a Particle-Size Reduction Apparatus

Protocol

The sterilisation protocol of the invention has been developed for aknown particle size reduction apparatus, namely a Microfluidics standardMF-210C Microfluidizer®, as part of a manufacturing process to providesterile Budesonide suspensions for Blow-Fill-Seal production ofnebulisation suspensions. The protocol is nevertheless believed to be ofapplication to suspensions of other drugs and also to particle-sizereduction using other equipment.

As an initial step, we demonstrated the ability to inactivate highlevels of contamination of an isolated intensifier and check valves, andthe following protocol was then developed for sterilisation of the wholeapparatus, to ensure that sterilising temperatures can be achievedthroughout the product contact areas and in the isolation chambers ofthe intensifier.

The protocol is designed to provide the temperatures and exposure timesrequired to achieve a minimum of 121° C. for 15 minutes, using eithersaturated steam, or superheated water under pressure, or both, and toprovide a 10⁶ reduction in G. stearothermophilus ATCC 7953 spores wheninoculated onto components of the Microfluidizer® considered likely torepresent the most difficult challenge to sterilisation. The protocol isdesigned to arrive at a set of operating conditions for sterilisation inplace using moist heat, employing either saturated steam or superheatedwater under pressure or both, which maintains at least 121° C. atinternal monitoring sites. The protocol may be modified and developed infuture to determine an adjusted minimum sterilising condition, includinga minimum sterilising time which in future sterilisation methods may beincreased to allow a margin of error in those methods.

This protocol covers the procedures to be followed during thesterilisation process. The apparatus is provided with a number ofpressure transducers, and Resistance Temperature Detectors (RTDs) fittedfor routine monitoring and during these studies the outputs of the RTDsand pressure transducers are fed to the validator (a Kaye Validator).Additional study thermocouples are positioned internally throughout theapparatus, wherever access is possible. Additional study thermocouplesmay be positioned externally to help indicate potential sites forroutine monitoring.

The equipment and materials used are Microfluidizer® Apparatus andServices, a Kaye Validator 2000, a Kaye Calibration Temperature sourceHTR400 or LTR140 or CTR40, or alternative equivalent provided by theapplicant and a Kaye IRTD calibration reference thermometer.

All critical operating instruments used in the sterilising procedurescovered by this protocol are calibrated, using standards traceable tonational standards. All critical test instruments used in the protocolare calibrated according to written procedures, using standardstraceable to national standards.

During the sterilisation process covered by this protocol, observationsof routine temperature and pressure indicators are made, and may bemodified prior to or during the studies to reflect the number of studytemperature and pressure test positions built into the apparatus forthese studies. Validation thermocouple data are automatically logged ata minimum frequency of every ten seconds from when heat is introducedinto the test apparatus.

Test temperature sensors and the data recorder are calibrated at 100° C.and 130° C., after the test thermocouples have stabilized to under 0.2°C. per minute for 5 minutes, with the reference thermometer stabilizedto within 0.012° C. during the final minute. Readings of each sensor andreference thermometer are taken at one-minute intervals for five minutesat each temperature point. Calibration of test sensors and data recorderis confirmed at 122° C. before and after qualification.

Temperatures derived from sensors and data recorder should not vary fromreference temperatures by more than ±0.5° C. Only thermocouples meetingthese criteria are used in the qualification.

A series of studies is conducted employing saturated steam orsuperheated water under pressure, to provide sterilising conditionsthroughout the product circuit and in the isolation chambers. Thepipework is adjusted to provide suitable services for the heat sourceemployed.

The studies are conducted over a range of temperatures and times (and,if necessary, pressures for the superheated water), until a suitable setof conditions is achieved which complies with the acceptance criteria.At least three consecutive acceptable studies are performed withunchanged sterilising settings before those settings can be consideredthe minimum suitable for further validation study.

Up to nine of the routine RTD temperature sensors supplied with theequipment and additional study thermocouples to a total of 36 sensors,including pressure transducers, are positioned in and on the apparatus.Internal thermocouples are introduced via appropriate Triclover® seals.External thermocouples may if desired be held in direct contact with thestainless steel surfaces.

Data collection commences when heat is applied to the product contactcircuit, and the isolation chamber, data being collected simultaneouslyfrom each temperature sensor, and each pressure sensor.

The time at which the first temperature probe reaches a minimum of 121°C., and at which all temperature probes reach 121° C. is recorded. Thetimed holding period commences when all test thermocouples have reached121° C., and continues until all test thermocouples have remained above121° C. continuously for a minimum of 15 minutes. At the end of theholding period, the apparatus is cooled. For steam sterilisations, theequipment is pressurised with air, and the steam pressure terminated.For superheated water sterilisations, the water in the circuit iscooled.

Results obtained from the above analyses must show compliance with thefollowing criteria for any set of sterilising conditions to beconsidered to provide minimum conditions for further study:

-   -   (1) All internal temperature test positions must record a        minimum of 121° C. continuously for at least the final 15        minutes of the holding period.    -   (2) For steam sterilisation, pressures measured must agree with        the saturated vapour pressure of steam at the temperature        measured at the same point, within ±1° C.

Results

18 steam sterilisation protocols were carried out according to theprotocol described above. The first run was a control (no spores) and inthe remaining 17 runs the following components of an M-210EHMicrofluidizer® were inoculated with 2×10⁶ heat resistant spores ofGeobacillus stearothermophilus ATCC No. 7953:

-   -   Runs 2-4 check valve spring retainer, intensifier plunger seal,        plunger contact sealing edge.    -   Runs 5-7 intensifier plunger seal, outer wall, behind barrel        contact sealing edge.    -   Runs 8-10 intensifier plunger seal, spring contact surface.    -   Runs 11-13 plunger bushing inner surface    -   Runs 14-16 plunger bushing outer surface, plastic seal support        ring, surface in contact with metal seal support ring.    -   Run 17 plunger bushing outer surface, plastic seal support ring,        surface in contact with metal seal support ring, PTFE        sealed-spring plunger seal.    -   Run 18 plunger bushing inner surface, plastic seal support ring,        surface in contact with metal seal support ring, Ultra High        Density PE sealed-spring plunger seal.

The steam sterilisation protocols were run to achieve 121° C. for 15minutes (as measured using a temperature probe embedded in oneintensifier barrel, close to the position of the plunger seal).

After this time, each inoculated component was then tested for sterilityaccording to Example 4 below. All components showed a 6-log reduction inheat-resistant spores—i.e. all components passed the sterility test (MCAguidelines).

Example 2 Validating Sterility of a Seal

A seal, which has previously been contaminated with at least 1×10⁶heat-resistant bacterial spores, is inserted into the bore of aparticle-size reduction apparatus. The particle-size reduction apparatusis sterilised as described in Example 1 above and then the seal isremoved from the apparatus. To validate the sterility of the apparatusbore, the seal is incubated with growth medium. A seal removed from anapparatus that has not undergone a sterilisation procedure is used as acontrol. The growth medium is examined for growth of microorganisms,which would indicate that the test seal (and hence the bore) had notbeen sterilised effectively. If there is no growth in the mediumcomprising the test seal, (growth being observed in the mediumcomprising the seal from the unsterilised bore) this indicates thatsterility is achieved.

Example 3 Reduction of Particle Size of a Sterile Suspension

The mass median diameter of particles of a Budesonide suspension isreduced using an M-210EH Microfluidizer® apparatus containing, a firstinteraction chamber having a single circular flow passage ofapproximately 400 μm diameter and a second interaction chamber having asingle circular flow passage of approximately 300 μm; that haspreviously been sterilised according to Example 1 above.

A sterile Budesonide suspension (12 litres), having particles of massmedian diameter approximately 50 μm is introduced into the sterileapparatus from the reaction feed tank. The pressure used isapproximately 34 MPa (5,000 psi) and the apparatus is run at 0.75 litresper minute. The suspension is passed through the apparatus and particlesize is monitored during each pass. After about 30 passes the massmedian diameter of particles in the suspension is reduced to 2-3 μm. Thesuspension is then transferred to a sterile packaging line for packaginginto sterile nebules.

Example 4 Particle-Size Distribution

The use of various different interaction chambers for comminution wasevaluated.

The protocol of Example 3 was repeated, however, the sterile Budesonidesuspension (12 litres) was passed through a sterilised M-210EHMicrofluidizer® apparatus, which had been modified to contain variousdifferent combinations of interaction chambers; i.e. interactionchambers provided with circular flow passages of diameter 200 μm, 250μm, 300 μm or 400 μm; with or without a first interaction chambercontaining a circular flow passage of 400 μm diameter.

In each batch of suspension processed, after each comminution cycle/passthe particle size of the suspension was monitored using an on-lineFocused Beam Reflectance Measurement (FBRM) probe, and a sample ofsuspension was taken for laser diffraction analysis.

The results from the laser diffraction studies are shown in Tables 1 and2, below. Table 1 demonstrates the particle size distribution (PSD) forthe final suspension of particles compared to the target particle sizedistribution profile. The target size distribution profile was thenchanged to reflect a suspension of smaller particles and the comminutionprocedure was carried out using more combinations of interactionchambers.

In each comminution procedure the number of cycles/passes through theMicrofluidizer® was increased or reduced until a PSD profile similar tothat of the target was achieved. TABLE 1 Results of laser diffractionanalysis with selected target PSD profile Flow passage diameter (μm)Batch 1^(st) interaction 2^(nd) interaction D10¹ D50² D90³ VMD⁴ No. ofNo. chamber chamber (μm) (μm) (μm) (μm) cycles Target 1 1-1.25 4.5-6.09.0-11.0 3.5-5.0 1 400 250 0.88 3.23 9.80 4.49 16 2 400 250 0.74 2.066.28 2.87 22 3 400 200 0.90 3.54 10.54 4.76 24¹10% of particles are of size given or smaller²50% of particles are of size given or smaller³90% of particles are of size given or smaller⁴Volume median diameter

TABLE 2 Results of laser diffraction analysis with selected target PSDprofile Flow passage diameter (μm) 1^(st) interaction 2^(nd) interactionD10¹ D50² D90³ VMD⁴ No. Batch No. chamber chamber (μm) (μm) (μm) (μm) ofcycles Target 2 0.79 2.48 6.98 3.29 4 400 200 0.73 2.06 6.28 2.87 30 5400 200 0.79 2.48 6.98 3.29 31 6 400 300 0.68 1.77 4.88 2.45 35 7 400300 0.71 2.01 6.35 2.85 30 8 300 — 0.76 2.27 6.46 3.02 20 9 300 — 0.832.52 6.65 3.21 17 10  300 — 0.78 2.29 6.44 3.02 19 11  300 — 0.81 2.346.20 2.99 16 12  300 — 0.80 2.30 6.17 2.97 16 13  300 — 0.78 2.17 5.822.81 16¹10% of particles are of size given or smaller²50% of particles are of size given or smaller³90% of particles are of size given or smaller⁴Volume median diameter

1. A method of producing a comminuted suspension of particles, whichcomprises: subjecting a suspension of particles to a comminutionprocedure carried out in a sterilised particle-size reduction apparatus;said particle size reduction apparatus comprising at least oneinteraction chamber for reducing the particle size of the suspension,each interaction chamber being provided with a flow passage throughwhich the suspension is forced, and an intensifier for forcing thesuspension through the flow passage of the interaction chamber orinteraction chambers, and recovering a suspension of particles ofreduced size; characterised in that the transverse cross-sectional areaof said flow passage is not less than 3.1×10⁴ μm².
 2. A method accordingto claim 1, wherein the transverse cross-sectional area of the flowpassage is in the range of 3.1×10⁴ to 2.8×10⁵ μm².
 3. A method accordingto claim 2, wherein the transverse cross-sectional area of the flowpassage is in the range of 4.9×10⁴ to 2.0×10⁵ μm².
 4. A method accordingto claim 3, wherein the transverse cross-sectional area of the flowpassage is in the range of 7.1×10⁴ to 1.3×10⁵ μm².
 5. A method accordingto claim 1, wherein the suspension is forced from the intensifier at apressure not exceeding 69 MPa (10,000 psi).
 6. A method according toclaim 5, wherein the suspension is forced from the intensifier at apressure in the range of 21-48 MPa (3,000-7,000 psi).
 7. A methodaccording to claim 6, wherein the suspension is forced from theintensifier at a pressure in the range of 28-41 MPa (4,000-6,000 psi).8. A method according to claim 7, wherein the suspension is forced fromthe intensifier at a pressure of approximately 34 Mpa (5,000 psi).
 9. Amethod according to claim 1, which comprises: (a) subjecting asuspension of particles to a comminution procedure carried out in asterilised particle-size reduction apparatus; said particle-sizereduction apparatus comprising at least one interaction chamber forreducing the particle size of the suspension, each interaction chamberbeing provided with a flow passage through which the suspension isforced, and an intensifier for forcing the suspension through the flowpassage of the interaction chamber or interaction chambers; to obtain acomminuted suspension of particles; characterised in that the transversecross-sectional area of said flow passage is not less than 3.1×10⁴ μm²;(b) optionally recovering a comminuted suspension of particles from step(a); (c) subjecting a comminuted suspension of particles from step (a)to at least one further comminution procedure carried out in asterilised particle-size reduction apparatus as defined in part (a); and(d) recovering a comminuted suspension of particles of reduced size. 10.A method according to claim 9, wherein up to 50 comminution proceduresare carried out.
 11. A method according to claim 10, wherein from 10 to50 comminution procedures are carried out.
 12. A method according toclaim 11, wherein from 14 to 40 comminution procedures are carried out.13. A method according to claim 12, wherein from 20 to 30 comminutionprocedures are carried out.
 14. A method according to claim 9, whereinstep (c) further comprises; recovering a comminuted suspension ofparticles after one or more further comminution procedures, measuringthe size of recovered particles, and on the basis of the measured sizes,subjecting the suspension to one or more further comminution procedures.15. A method according to claim 1, wherein the mass median diameter ofparticles in the recovered suspension of particles is in the range of1-10 μm.
 16. A method according to claim 15, wherein the mass mediandiameter of particles is in the range of 2-3 μm.
 17. A method accordingto claim 1, wherein the particles comprise Budesonide or Fluticasone.18. A method according to claim 1, further comprising the step ofpackaging the suspension into sterile ampoules.
 19. A nebule containinga sterile suspension prepared according to claim
 15. 20. (canceled) 21.A sterilisable particle-size reduction apparatus, comprising: at leastone interaction chamber for reducing the particle size of thesuspension, each interaction chamber being provided with a flow passagethrough which the suspension is forced; and an intensifier for forcingthe suspension through the flow passage of the interaction chamber orinteraction chambers; characterised in that the transversecross-sectional area of said flow passage is not less than 3.1×10⁴ μm².22. A sterilisable particle-size reduction apparatus according to claim21, wherein the transverse cross-sectional area of the flow passage isin the range of 3.1×10⁴ to 2.8×10⁵ μm².
 23. A sterilisable particle-sizereduction apparatus according to claim 22, wherein the transversecross-sectional area of the flow passage is in the range of 4.9×10⁴ to2.0×10⁵ μm².
 24. A sterilisable particle-size reduction apparatusaccording to claim 23, wherein the transverse cross-sectional area ofthe flow passage is in the range of 7.1×10⁴ to 1.3×10⁵ μm².
 25. Asterilisable particle-size reduction apparatus according to claim 21,which comprises from 1 to 4 interaction chambers arranged in series. 26.A sterilisable particle-size reduction apparatus according to claim 25,which comprises a first and a second interaction chamber arranged inseries.
 27. A sterilisable particle-size reduction apparatus accordingto claim 26, wherein said first and second interaction chambers are eachprovided with a single flow passage.
 28. A sterilisable particle-sizereduction apparatus according to claim 27, wherein the transversecross-sectional area of the flow passage of said first interactionchamber is approximately the same as the transverse cross-sectional areaof the flow passage of said second interaction chamber.
 29. Asterilisable particle-size reduction apparatus according to claim 27,wherein the transverse cross-sectional area of the flow passage of saidfirst interaction chamber is greater than the transverse cross-sectionalarea of the flow passage of said second interaction chamber.
 30. Asterilisable particle-size reduction apparatus according to claim 29,wherein: the transverse cross-sectional area of the flow passage of saidfirst interaction chamber is approximately 1.3×10⁵ μm², and thetransverse cross-sectional area of the flow passage of said secondinteraction chamber is approximately 7.1×10⁴ μm².
 31. A sterilisableparticle-size reduction apparatus according to claim 21, furthercomprising a heat exchanger to maintain the temperature of thesuspension at from 7° C. to 40° C. in use.
 32. A sterilisableparticle-size reduction apparatus according to claim 21, furthercomprising a pressure relief valve which is a rupture disc.
 33. Asterilisable particle-size reduction apparatus according to claim 21,wherein the intensifier is connected to the interaction chamber orchambers by one or more conduits, each conduit being provided with anon-return valve having metal-to-metal seats.
 34. A sterilisableparticle-size reduction apparatus according to claim 21, furthercomprising a first heat exchanger to maintain the temperature of thesuspension in the interaction chamber and a second heat exchanger tomaintain the temperature of the suspension in the intensifier, whereinthe first and second heat exchangers are independently controlled.
 35. Asterilisable particle-size reduction apparatus according to claim 21,wherein the intensifier is driven by a working fluid and the apparatusfurther comprises a seal that prevents the suspension from reaching theworking fluid in the event of failure of a plunger seal in theintensifier.
 36. A method of sterilising a particle-size reductionapparatus, comprising charging a non-sterile particle size reductionapparatus according to claim 21 with steam, to achieve sterilisation.37. A method according to claim 36, comprising validating saidsterilisation by: (a) under sterile conditions, removing a component ofsaid particle-size reduction apparatus; (b) under sterile conditions,transferring the component to growth medium; and (c) observing whetherthere is growth of microorganisms in the growth medium, therebydetermining whether the component is sterile.
 38. A method according toclaim 36, comprising insulating valves and conduits downstream of theinteraction chamber, so as to maintain steam temperature duringsterilisation.
 39. A method according to claim 36, comprising:connecting steam traps to the apparatus; connecting temperature monitorsto the apparatus; introducing steam into the apparatus; monitoringtemperature at each of the temperature monitors; noting the time atwhich temperature recorded by each of the temperature monitors hasreached the sterilising temperature; continuing to introduce steam intothe apparatus for a predetermined period.
 40. A method according toclaim 39, wherein the period is determined by: introducingheat-resistant bacterial spores into the apparatus; introducing steaminto the apparatus and monitoring apparatus temperature until it hasreached the sterilising temperature; continuing to introduce steam for afirst known amount of time; determining whether after that first knownamount of time sterilisation has been achieved; and if sterilisation hasnot been achieved, repeating the method for a second, longer knownamount of time.
 41. A method according to claim 40, whereinsterilisation is deemed to have occurred when a six-log reduction inheat-resistant bacterial spores has been achieved.
 42. A methodaccording to claim 39, wherein the sterilising temperature is 121° C.43. A method according to claim 36, comprising introducing steam intothe intensifier and downstream of the interaction chambers.
 44. A methodaccording to claim 36, comprising pre-heating the interaction chambers.45. A method according to claim 36, wherein all steam exiting theintensifier passes through the interaction chambers.
 46. A method ofsterilising a particle-size reduction apparatus, comprising charging anon-sterile particle size reduction apparatus according to claim 21 withpressurised, superheated water so as to sterilise the apparatus.
 47. Amethod according to claim 46, comprising operating the intensifier so asto control the temperature of the water during sterilisation.
 48. Amethod according to claim 47, comprising adjusting pressure within theapparatus so as to adjust temperature within the apparatus.
 49. A methodaccording to claim 46, comprising introducing the water into theintensifier and introducing steam into the isolation area of theintensifier, the steam being at the same temperature as or at a highertemperature than the water.
 50. A method according to claim 49, whereinthe steam is at a temperature at least 0.5° C. higher than the water.51. A method according to claim 46, comprising: introducing water at atemperature below 100° C. into the apparatus; and operating theapparatus so as to increase the temperature of the water to thesterilising temperature.
 52. A method according to claim 51, wherein thesterilising temperature is 121° C.
 53. A method according to claim 46,comprising flushing the apparatus with sterile air once the apparatushas been sterilised.
 54. A method according to claim 46, wherein thepressurised, superheated water comprises a surfactant.
 55. A method ofpreparing a sterile suspension, comprising: obtaining a sterileparticle-size reduction apparatus according to claim 21; passing asterile suspension through the sterile apparatus; and monitoringparticle size in the suspension.
 56. A method according to claim 55,wherein said sterile particle-size reduction apparatus has beensterilized by charging with steam to achieve sterilisation.
 57. A methodaccording to claim 55, wherein the sterile suspension comprisesBudesonide or Fluticasone.
 58. A method according to claim 55, whereinparticle size is monitored until a final particle size in the suspensionof mass median diameter 2-3 μm is obtained.
 59. A method according toclaim 55, further comprising the step of packaging the sterilesuspension into sterile ampoules.
 60. A nebule containing a sterilesuspension prepared according to claim
 55. 61. A method of validatingthe sterility of components of a particle-size reduction apparatusaccording to claim 21, comprising the steps of: (a) under sterileconditions, removing a component of said particle-size reductionapparatus; (b) under sterile conditions, transferring the component togrowth medium; and (c) observing whether there is growth ofmicroorganisms in the growth medium, thereby determining whether thecomponent is sterile.
 62. A method according to claim 61, furthercomprising the initial steps of: inoculating a component of saidparticle-size reduction apparatus with heat-resistant bacterial spores;inserting the component into the particle-size reduction apparatus; andcarrying out a sterilisation by charging said particle-size reductionapparatus with steam to achieve sterilisation.
 63. A method according toclaim 62, wherein said component is inoculated with at least 1×10⁶heat-resistant bacterial spores.
 64. A modification to the methodclaimed in claim 1, wherein the interaction chamber and intensifier areintegrally combined into a pump.